This invention relates to four chimeric genes, a first encoding lysine-insensitive aspartokinase (AK), which is operably linked to a plant chloroplast transit sequence, a second encoding lysine-insensitive dihydrodipicolinic acid synthase (DHDPS), which is operably linked to a plant chloroplast transit sequence, a third encoding a lysine-rich protein, and a fourth encoding a plant lysine ketoglutarate reductase, all operably linked to plant seed-specific regulatory sequences. Methods for their use to produce increased levels of lysine or threonine in the seeds of transformed plants are provided. Also provided are transformed plants wherein the seeds accumulate lysine or threonine to higher levels than untransformed plants.
Human food and animal feed derived from many grains are deficient in some of the ten essential amino acids which are required in the animal diet. In corn (Zea mays L.), lysine is the most limiting amino acid for the dietary requirements of many animals. Soybean (Glycine max L.) meal is used as an additive to corn based animal feeds primarily as a lysine supplement. Thus an increase in the lysine content of either corn or soybean would reduce or eliminate the need to supplement mixed grain feeds with lysine produced via fermentation of microbes.
Plant breeders have long been interested in using naturally occuring variations to improve protein quality and quantity in crop plants. Maize lines containing higher than normal levels of lysine (70%) have been identified [Mertz et al. (1964) Science 145:279, Mertz et al. (1965) Science 150:1469-70]. However, these lines which incorporate a mutant gene, opaque-2, exhibit poor agronomic qualities (increased susceptibility to disease and pests, 8-14% reduction in yield, low kernel weight, slower drying, lower dry milling yield of flaking grits, and increased storage problems) and thus are not commercially useful [Deutscher (1978) Adv. Exp. Medicine and Biology 105:281-300]. Quality Protein Maize (QPM) bred at CIMMYT using the opaque-2 and sugary-2 genes and associated modifiers has a hard endosperm and enriched levels of lysine and tryptophan in the kernels [Vasal, S. K., et al. Proceedings of the 3rd seed protein symposium, Gatersleben, Aug. 31-Sep. 2, 1983]. However, the gene pools represented in the QPM lines are tropical and subtropical. Quality Protein Maize is a genetically complex trait and the existing lines are not easily adapted to the dent germplasm in use in the United States, preventing the adoption of QPM by corn breeders.
The amino acid content of seeds is determined primarily (90-99%) by the amino acid composition of the proteins in the seed and to a lesser extent (1-10%) by the free amino acid pools. The quantity of total protein in seeds varies from about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes. Much of the protein-bound amino acids is contained in the seed storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. In many seeds the storage proteins account for 50% or more of the total protein.
To improve the amino acid composition of seeds genetic engineering technology is being used to isolate, and express genes for storage proteins in transgenic plants. For example, a gene from Brazil nut for a seed 2S albumin composed of 26% sulfur-containing amino acids has been isolated [Altenbach et al. (1987) Plant Mol. Biol. 8:239-250] and expressed in the seeds of transformed tobacco under the control of the regulatory sequences from a bean phaseolin storage protein gene. The accumulation of the sulfur-rich protein in the tobacco seeds resulted in an up to 30% increase in the level of methionine in the seeds [Altenbach et al. (1989) Plant Mol. Biol. 13:513-522]. However, no plant seed storage proteins similarly enriched in lysine relative to average lysine content of plant proteins have been identified to date, preventing this approach from being used to increase lysine.
An alternative approach is to increase the production and accumulation of specific free amino acids such as lysine via genetic engineering technology. However, little guidance is available on the control of the biosynthesis and metabolism of lysine in the seeds of plants.
Lysine, along with threonine, methionine and isoleucine, are amino acids derived from aspartate, and regulation of the biosynthesis of each member of this family is interconnected. Regulation of the metabolic flow in the pathway appears to be primarily via end products. The first step in the pathway is the phosphorylation of aspartate by the enzyme aspartokinase (AK), and this enzyme has been found to be an important target for regulation in many organisms. However, detailed physiological studies on the flux of 4-carbon molecules through the aspartate pathway have been carried out in the model plant system Lemna paucicostata [Giovanelli et al. (1989) Plant Physiol. 90:1584-1599]. The authors state xe2x80x9cThese data now provide definitive evidence that the step catalyzed by aspartokinase is not normally an important site for regulation of the entry of 4-carbon units into the aspartate family of amino acids [in plants].xe2x80x9d
The aspartate family pathway is also believed to be regulated at the branch-point reactions. For lysine this is the condensation of aspartyl xcex2-semialdehyde with pyruvate catalyzed by dihydrodipicolinic acid synthase (DHDPS), while for threonine and methionine the reduction of aspartyl xcex2-semialdehyde by homoserine dehydrogenase (HDH) followed by the phosphorylation of homoserine by homoserine kinase (HK) are important points of control.
The E. coli dapA gene encodes a DHDPS enzyme that is about 20-fold less sensitive to inhibition by lysine than than a typical plant DHDPS enzyme, e.g., wheat germ DHDPS. The E. coli dapA gene has been linked to the 35S promoter of Cauliflower Mosaic Virus and a plant chloroplast transit sequence. The chimeric gene was introduced into tobacco cells via transformation and shown to cause a substantial increase in free lysine levels in leaves [Glassman et al. (1989) PCT Patent Appl. PCT/US89/01309, Shaul et al. (1992) Plant Jour. 2:203-209, Galili et al. (1992) EPO Patent Appl. 91119328.2]. However, the lysine content of the seeds was not increased in any of the transformed plants described in these studies. The same chimeric gene was also introduced into potato cells and lead to small increases in free lysine in leaves, roots and tubers of regenerated plants [Galili et al. (1992) EPO Patent Appl. 91119328.2, Perl et al. (1992) Plant Mol. Biol. 19:815-823]. These workers have also reported on the introduction of an E. coli lysC gene that encodes a lysine-insensitive AK enzyme into tobacco cells via transformation [Galili et al. (1992) Eur. Patent Appl. 91119328.2; Shaul et al. (1992) Plant Physiol. 100:1157-1163]. Expression of the E. coli enzyme results in increases in the levels of free threonine in the leaves and seeds of transformed plants. Crosses of plants expressing E. coli DHDPS and AK resulted in progeny that accumulated more free lysine in leaves than the parental DHDPS plant, but less free threonine in leaves than the parental AK plant. No evidence for increased levels of free lysine in seeds was presented.
The limited understanding of the details of the regulation of the biosynthetic pathway in plants makes the application of genetic engineering technology, particularly to seeds, uncertain. There is little information available on the source of the aspartate-derived amino acids in seeds. It is not known, for example, whether they are synthesized in seeds, or transported to the seeds from leaves, or both, from most plants. In addition, free amino acids make up only a small fraction of the total amino acid content of seeds. Therefore, over-accumulation of free amino acids must be many-fold in order to significantly affect the total amino acid composition of the seeds. Furthermore, little is known about catabolism of free amino acids in seeds. Catabolism of free lysine has been observed in developing endosperm of corn and barley. The first step in the catabolism of lysine is believed to be catalyzed by lysine-ketoglutarate reductase [Brochetto-Braga et al. (1992) Plant Physiol. 98:1139-1147]. Whether such catabolic pathways are widespread in plants and whether they affect the level of accumulation of free amino acids is unknown. Finally, the effects of over-accumulation of a free amino acid such as lysine or threonine on seed development and viability is not known.
Before this patent application no method to increase the level of lysine or threonine, or any other amino acid, in seeds via genetic engineering was known. Furthermore, no examples of seeds having increased lysine or threonine levels obtained via genetic engineering were known before the invention described herein. Thus, there is a need for genes, chimeric genes, and methods for expressing them in seeds so that an over-accumulation of free amino acids in seeds will result in an improvement in nutritional quality.
This invention concerns an isolated nucleic acid fragment comprising:
(a) a first nucleic acid subfragment encoding apartokinase which is substantially insensitive to inhibition by lysine; and
(b) a second nucleic acid subfragment encoding dihydrodipicolinic acid synthase which is substantially insensitive to inhibition by lysine.
The invention also concerns an isolated nucleic acid fragment comprising a nucleic acid subfragment encoding lysine ketoglutarate reductase.
Further disclosed herein is a nucleic acid fragment comprising
(a) a first chimeric gene wherein a nucleic acid fragment encoding aspartokinase which is substantially insensitive to inhibition by lysine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(b) a second chimeric gene wherein a nucleic acid fragment encoding dihydrodipicolinic acid synthase which is substantially insensitive to inhibition by lysine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence.
Additionally disclosed is an isolated nucleic acid fragment comprising:
(a) a first chimeric gene wherein a nucleic acid fragment comprising a nucleotide sequence essentially similar to the sequence shown in SEQ ID NO:1: encoding E. coli AKIII, said nucleic acid fragment encoding a lysine-insensitive variant of E. coli AKIII and further characterized in that at least one of the following conditions is met:
(1) the amino acid at position 318 is an amino acid other than threonine, or
(2) the amino acid at position 352 is an amino acid other than methionine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence and
(b) a second chimeric gene wherein a nucleic acid fragment derived from a bacteria encoding dihydrodipicolinic acid synthase is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(c) a third chimeric gene wherein a nucleic acid fragment encoding part or all of lysine ketoglutarate reductase is operably linked in the sense or antisense orientation to a seed-specific regulatory sequence.
Also disclosed is an isolated nucleic acid fragment comprising at least one nucleotide sequence essentially similar to the sequence shown in SEQ ID NO:1 encoding E. coli AKIII, said nucleic acid fragment encoding a lysine-insensitive variant of E. coli AKIII and further characterized in that at least one of the following conditions is met:
(a) the amino acid at position 318 is an amino acid other than threonine, or
(b) the amino acid at position 352 is an amino acid other than methionine.
Also claimed is an embodiment wherein the nucleic acid fragment discussed herein is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence.
Plants and seeds comprising in their genomes the described nucleic acid fragments and/or genes are also disclosed.
The invention also concerns a method for increasing the threonine content of the seeds of plants, and plants produced by such method wherein the plant is capable of transmitting said chimeric gene to a progeny plant and wherein the progeny plant has the ability to produce levels of free threonine at least two times greater than the free threonine levels of untransformed plants, which method comprises:
(a) transforming plant cells with the above described chimeric gene;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds; and
(c) selecting from the progeny seed of step (b) for those seeds containing increased levels of threonine.
Also described is a method for increasing the lysine content of the seeds of plants and plants produced by such methods wherein the plant is capable of transmitting said nucleic acid fragment to a progeny plant and wherein the progeny plant has the ability to produce levels of free lysine at least two times greater than free lysine levels of plants not containing the nucleic acid fragment, which method comprises:
(a) transforming plant cells with the above described nucleic acid fragments;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds; and
(c) selecting from the progeny seed of step (b) those seeds containing increased levels of lysine.
Further disclosed is an isolated nucleic acid fragment comprising:
(a) a first nucleic acid subfragment encoding apartokinase which is substantially insensitive to inhibition by lysine; and
(b) a second nucleic acid subfragment encoding dihydrodipicolinic acid synthase which is substantially insensitive to inhibition by lysine; and
(c) a third nucleic acid subfragment encoding a lysine-rich protein wherein the weight percent lysine is at least 15%.
Also disclosed herein are an isolated nucleic acid fragment comprising:
(a) a first chimeric gene wherein a nucleic acid fragment comprising a nucleotide sequence essentially similar to the sequence shown in SEQ ID NO:1: encoding E. coli AKIII, said nucleic acid fragment encoding a lysine-insensitive variant of E. coli AKIII and further characterized in that at least one of the following conditions is met:
(1) the amino acid at position 318 is an amino acid other than threonine, or
(2) the amino acid at position 352 is an amino acid other than methionine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence and
(b) a second chimeric gene wherein a nucleic acid fragment derived from a bacteria encoding dihydrodipicolinic acid synthase is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(c) a third chimeric gene wherein a nucleic acid fragment encoding a lysine-rich protein wherein the weight percent lysine is at least 15% is operably linked to a seed-specific regulatory sequence.
Further disclosed herein is an isolated nucleic acid fragment, and plants and seeds containing such fragment, comprising:
(a) a first chimeric gene wherein a nucleic acid fragment comprising a nucleotide sequence essentially similar to the sequence shown in SEQ ID NO:1: encoding E. coli AKIII, said nucleic acid fragment encoding a lysine-insensitive variant of E. coli AKIII and further characterized in that at least one of the following conditions is met:
(1) the amino acid at position 318 is an amino acid other than threonine, or
(2) the amino acid at position 352 is an amino acid other than methionine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence and
(b) a second chimeric gene wherein a nucleic acid fragment derived from a bacteria encoding dihydrodipicolinic acid synthase is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(c) a third chimeric gene wherein a nucleic acid fragment encoding a lysine-rich protein comprising n heptad units (d e f g a b c), each heptad being either the same or different, wherein:
n is at least 4;
a and d are independently selected from the group consisting of Met, Leu, Val, Ile and Thr;
e and g are independently selected from the group consisting of the acid/base pairs Glu/Lys, Lys/Glu, Arg/Glu, Arg/Asp, Lys/Asp, Glu/Arg, Asp/Arg and Asp/Lys; and
b, c and f are independently any amino acids except Gly or Pro and at least two amino acids of b, c and f in each heptad are selected from the group consisting of Glu, Lys, Asp, Arg, His, Thr, Ser, Asn, Ala, Gln and Cys,
said nucleic acid fragment is operably linked to a seed-specific regulatory sequence.
Further disclosed herein is an isolated nucleic acid fragment, and plants and seeds containing such fragment, comprising:
(a) a first chimeric gene wherein a nucleic acid fragment comprising a nucleotide sequence essentially similar to the sequence shown in SEQ ID NO:1: encoding E. coli AKIII, said nucleic acid fragment encoding a lysine-insensitive variant of E. coli AKIII and further characterized in that at least one of the following conditions is met:
(1) the amino acid at position 318 is an amino acid other than threonine, or
(2) the amino acid at position 352 is an amino acid other than methionine is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(b) a second chimeric gene wherein a nucleic acid fragment derived from a bacteria encoding dihydrodipicolinic acid synthase is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence; and
(c) a third chimeric gene wherein a nucleic acid fragment encoding a lysine-rich protein having the amino acid sequence (MEEKLKA)6(MEEKMKA)2 is operably linked to a seed-specific regulatory sequence.
Also disclosed are plants comprising in their genome the nucleic acid fragments listed above, plants comprising in their genomes each of the chimeric genes discribed above and seeds obtained from such plants.
Also disclosed is a method for increasing the lysine content of the seeds of plants comprising:
(a) transforming plant cells with the nucleic acid fragment listed above;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds; and
(c) selecting from the progeny seed of step (b) those seeds containing increased levels of lysine.
The invention also includes a novel transformed plant, preferably a rapeseed or soybean plant, wherein the seeds of the plant accumulate lysine at a level at least ten percent higher than do seeds of an untransformed plant (10% to 400% higher for soybean and 10% to 100% higher for rapeseed) than do seeds of an untransformed plant.
Further disclosed herein is a nucleic acid acid fragment wherein the seed-specific regulatory sequence is a monocot embyro-specific promoter, a monocot plant comprising in its genome such nucleic acid fragment and a seed obtained from that plant and comprising in its genome that nucleic acid fragment.
Disclosed is a method for increasing the lysine content of the seeds of monocot plants comprising:
(a) transforming plant cells with the nucleic acid fragment of claim 33;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds; and
(c) selecting from the progeny seed of step (b) those seeds containing increased levels of lysine, and plants produced by such a method, wherein the plant is capable of transmitting said nucleic acid fragment to a progeny plant and wherein the progeny plant has the ability to produce levels of free lysine at least five times greater than free lysine levels of plants not containing the nucleic acid fragment.
Also disclosed is a transformed corn plant wherein the seeds of the plant accumulate lysine to a level between ten percent and one hundred thirty percent higher than do seeds of an untransformed plant and a method for increasing the lysine content and reducing the accumulation of lysine breakdown products of the seeds of plants.
Further disclosed is a method for increasing the lysine content and reducing the accumulation of lysine breakdown products of the seeds of plants comprising:
(a) transforming plant cells with the nucleic acid fragment described herein, then
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
(c) selecting from the progeny seed of step (b) those seeds containing increased levels of lysine; and lysine breakdown products and
(d) introducing a mutation in the gene encoding lysine ketoglutarate reductase which reduces the enzyme activity and reduces accumulation of lysine breakdown products.